Over the last 10 to 15 years, mass spectrometric analysis has successfully used matrix assisted laser desorption/ionization (MALDI) for the ionization of biological macromolecules, in particular proteins or peptides.
In matrix assisted laser desorption/ionization, analyte molecules are embedded in a matrix layer. The prepared MALDI sample is irradiated with a short laser pulse that is strongly absorbed by the matrix layer. The pulsed irradiation explosively converts the matrix substance from the solid phase to the gas phase, creating a vaporization cloud (desorption). The analyte molecules are generally ionized by protonation or deprotonation in reactions with the molecules or ions of the matrix substance. The analyte ions are predominantly singly charged after they leave the vaporization cloud.
More than a hundred different chemical matrix substances are known for analyte molecules of different classes of chemical substances, such as proteins or nucleic acids. These include sinapic acid, DHB (i.e., 2,5-dihydroxy-benzoic acid), CHCA (i.e., α-cyano-4-hydroxy cinnamic acid) and HPA (i.e., 3-hydroxypicolinic acid). Only about half a dozen matrix substances have become widely used; different matrix substances have been found optimal for different analytical tasks.
Homogeneous biological samples, such as tissue homogenates, lyzed bacteria or biological fluids (e.g., urine, blood serum, lymph, spinal fluid, tears, sputum) are prepared using a variety of methods, such as “dried droplet” preparation or thin layer preparation. Preparation on the sample support is often preceded by chromatographic or electrophoretic fractionation.
In dried droplet preparation, a matrix solution is pipetted onto a sample support together with analyte molecules, and then dried. The matrix substance crystallizes, and the analyte molecules, present at extremely low concentrations, are embedded in the crystals of the matrix layer, or at their grain boundaries as individual molecules, separated from each other. In thin layer preparation, on the other hand, a suitable matrix solution without any analyte molecules is first pipetted onto the sample support, and a thin, microcrystalline matrix layer is created by quickly evaporating the solvent. A solution containing analyte molecules is then applied to the thin, microcrystalline matrix layer and dried; the sample prepared in this way may, optionally, be washed either during or after the drying process. In both dried droplet preparation and thin layer preparation, the matrix layer can, optionally, be recrystallized by the addition of a suitable solvent.
Imaging mass spectroscopy (IMS) is most often used to examine thin tissue sections rather than homogeneous biological samples. A thin tissue section is prepared, for instance, from a frozen tissue sample taken from a human, animal, or plant organ of interest using a cryomicrotome. It is then placed on an electrically conductive sample support, usually consisting of a glass specimen slide with a transparent conductive coating. A matrix solution is applied to the thin tissue section using an appropriate method. After the matrix layer has dried, the sample slide is inserted directly into the mass spectrometer. The raster scan method according U.S. Pat. No. 5,808,300 to Caprioli or stigmatic imaging of the ions of a small region of the sample as disclosed by S. L. Luxembourg et al., Anal. Chem. 2003; 75, 2333-41 may be used for the subsequent mass spectrometric examination. In addition to thin tissue sections, carriers from thin layer chromatography, gel-electrophoretic membranes or blot membranes may also be considered as samples for imaging mass spectrometric analysis. In these samples, the analyte molecules are already located on the sample support prior to preparation of the matrix layer.
A variety of methods for the preparation of matrix layers for imaging mass spectrometric analysis are known from German patent application DE 10 2006 019 530.2. The matrix solution, or a recrystallization solution, can be applied to the sample by pneumatic spraying, vibration nebulizing or by nanospotting of droplets. The application of the matrix solution is not trivial, because (a) lateral smearing of the analyte molecules must be avoided, (b) the analyte molecules must, as far as possible, be extracted from the sample and incorporated into the crystals of the matrix layer, and (c) a favorable ratio of analyte molecules to contaminants must be achieved.
It has been found that when preparing the matrix layer for imaging mass spectrometric analysis, the matrix solution is favorably applied in the course of several cycles. A large number of individual droplets are applied in each cycle, but not so many that the droplets flow together on the surface to form a liquid film. How much matrix solution is applied, how often, what incubation and drying times are selected, and how fast the drying rate is, are all extremely important for the quality of samples prepared for imaging mass spectrometric analysis. Since little equipment for these procedures is available commercially, they are mostly carried out manually, with the result that only very limited reproducibility is possible. But even when automatic machines are used to apply the matrix solution, important parameters such as the rate of gas flow, the temperature during drying, the proportion of solvent in the air, or the temperature of the sample carrier are generally not held constant.
For these reasons, the preparation of a matrix layer for homogeneous samples is often not adequately reproducible.
A quantitative assessment of the quality of prepared samples has in the past been carried out exclusively by a mass spectrometric control measurement of a known reference substance, in which the presence and intensities (signal strengths) of specified ion signals in the measured mass spectrum are used as a quality criterion.
The preparation of the matrix layer comprises the application and drying of solutions, such as a matrix solution or a recrystallization solution. The recrystallization solution partially dissolves a matrix layer that is already present on the sample carrier. After the drying, analyte molecules from the existing matrix layer, or analyte molecules from the recrystallization solution, are embedded in the prepared matrix layer. If the matrix solution is nebulized to create droplets for application, these droplets may dry when they are still above the sample support, causing a fine “snow” of crystals to land on the sample support. The matrix layer is then prepared by applying a recrystallization solution.